Obtaining nucleic acid sequence information is an important starting point for medical and academic research endeavors. The sequence information facilitates medical studies of active disease, genetic disease predispositions, and assists in rational design of drugs targeting specific diseases. Sequence information is also the basis for genomic and evolutionary studies, and many genetic engineering applications. Reliable sequence information is critical for paternity tests, criminal investigations, and forensic studies.
Nucleic acid sequence information is typically obtained using chain termination and size separation procedures, such as those described by Sanger, et al., (1977 Proc. Nat. Acad. Sci. USA 74:5463-5467). Prior to gel separation, the nucleic acid target molecules of interest are cloned, amplified, and isolated. Then the sequencing reactions are conducted in four separate reaction vessels, one for each nucleotide: A, G, C and T. These sequencing methods are adequate for read lengths of 500-10000 nucleotides. However, they are time-consuming and require relatively large amounts of target molecules. Additionally, these methods can be expensive, as they require reagents for four reaction vessels. And the amplification steps are error-prone which can jeopardize acquiring reliable sequence information. Furthermore, these methods suffer from sequence-dependent artifacts including band compression during size separation.
The technological advances in automated sequencing machines, fluorescently-labeled nucleotides, and detector systems, have improved the read lengths, and permit massively parallel sequencing runs for high throughput methods. But these procedures are still inadequate for large projects, like sequencing the human genome. The human genome contains approximately three billion bases of DNA sequence. Procedures that can sequence and analyze the human genome (or the genome of any organism) in a relatively short time span and at a reduced cost will make it feasible to deliver genomic information as part of a healthcare program which can prevent, diagnose, and treat disease.
The energy transfer system provided herein overcomes many problems associated with current nucleotide incorporation procedures. The energy transfer system requires minute amounts of target molecule with no amplification steps, there is no need to perform four separate nucleotide incorporation reactions, and the reactions are not size separated or loaded on a gel. The energy transfer system is a single molecule sequencing system which facilitates rapid, accurate, and real-time sequencing of long nucleic acid fragments.